The ability to degrade proteins is an essential function of all eukaryotic cells. The ubiquitin-proteasome system has evolved to play an active role in cellular quality control by selective degradation of normal or damaged proteins. The ubiquitin-proteasome system is fundamental to cell cycle control, transcriptional regulation, stress response, immune and inflammatory responses and other vital processes. (For a review of the ubiquitin-proteasome system, see Hershko and Ciechanover, 1998, Annu. Rev. Biochem, 67:425-479; Varshaysky, 1997, Trends Biochem. Sci., 22: 383-387; and Hochstrasser, 1996, Annu. Rev. Genet, 30: 405-439.)
Ubiquitin (Ub) is a highly conserved 76-amino acid protein. Protein degradation via the ubiquitin-proteasome pathway generally involves covalent attachment of multiple molecules of ubiquitin to the protein substrate. The protein substrate is subsequently degraded by the 26S proteasome complex, and the free ubiquitin is recycled. There are also examples of proteins whose functions appear to be regulated by ubiquitylation, although ubiquitylation does not appear to target them for degradation (Hwang et al., 2003, Mol. Cell, 11: 261-266).
The attachment of ubiquitin to many known substrate proteins is believed to occur in a series of enzymatic reactions carried out sequentially by three classes of proteins: an ubiquitin-activating enzyme (E1) activates ubiquitin in an ATP-dependent manner to form a thioester bond between the carboxy-terminal Gly of Ub and a Cys residue of E1; activated Ub is then transferred to an ubiquitin-conjugating enzyme (E2 or UBC) to form another thioester bond; and a ubiquitin ligase (E3) catalyzes or promotes, in a substrate specific manner, the transfer of Ub from the E2 to a Lys residue of the substrate protein to form an isopeptide bond. An internal Lys residue of Ub can also form an isopeptide bond with the C-terminus of another Ub to create a multi-Ub chain that serves as a targeting signal for proteasome.
One specific example of an important ubiquitylation pathway is N-end rule ubiquitylation, and especially N-end rule ubiquitylation where ubiquitylation is preceded by N-terminal segment cleavage, where the N-terminal segment comprises one or more amino acid residues. The proteolysis exposes an N-degron, which comprises a destabilizing N-terminal residue plus an internal Lys residue where a multi-Ub chain is later attached. The N-terminal segment is cleaved to form an activated substrate of the ubiquitin-dependent N-end rule pathway (activated fragment), which is recognized through the exposed destabilizing N-terminal residue.
Prior to the discovery of the new N-terminal acetylation (Nt-acetylation) branch of the N-end rule pathway has been the subject of several review articles (Varshaysky, 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 12142). The ubiquitin ligase UBR1, an E3 ligase, has been shown to ubiquitylate N-end rule substrates and has two binding sites for primary destabilizing N-terminal residues. The type I site is specific for basic N-terminal residues Arg, Lys, His. The type II site is specific for bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Dipeptides carrying type I or type II N-terminal residues can serve as inhibitors of ubiquitylation of the corresponding type I or type II N-end rule substrates (Gonda et al., 1989, J. Biol. Chem., 264: 16700-16712). UBR1 from yeast contains yet another substrate-binding site, which recognizes proteins for ubiquitylation through an internal recognition site on substrates; this process can be enhanced by the presence of type I and type II dipeptides (Turner et al., 2000, Nature, 405: 579-583).
The degradation signal for ubiquitylation via the N-end rule pathways is termed an “N-degron” and comprises the primary destabilizing N-terminal residue and an internal lysine which is the site of ubiqutylation. Destabilizing N-terminal residues can be generated through proteolytic cleavages of specific proteins and other N-terminal modifications which reveal destabilizing residues at the new N-terminus. The residues that are exposed or modified to reveal an N-degron have been termed a “pre-N-degron” or “pro-N-degron.” For example, Sindbis virus RNA polymerase is produced during viral infection through site-specific cleavage of the viral polyprotein precursor and carries an N-terminal Tyr that has been shown in rabbit reticulocyte lysates to target the protein for ubiquitylation via the N-end rule pathway (deGroot et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88: 8967-8971). Another example is RGS4, whose N-terminal degradation signal is generated through a series of N-terminal modifications including (i) removal of N-terminal Met and exposure of Cys-2 at the N-terminus, (ii) oxidation of Cys-2 into cysteic acid, and (iii) conjugation of Arg to the N-terminus of the protein (Kwon et al., 2002, Science, 297: 96-99).
Relatively few physiological N-end rule substrates have been characterized to date. However, recently discovered N-end rule substrates linked to disease or pathology demonstrate the biological importance of N-end rule ubiquitylation pathway. For example, a carboxy-terminal fragment of cohesin in Saccharomyces cerevisiae is a physiological substrate for the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of this fragment is lethal and, in cells that lack an N-end rule ubiquitylation pathway, a highly increased frequency of chromosome loss is detected (Rao et al., 2001, Nature, 410: 955-959). Recent studies also indicate that enhanced protein breakdown in skeletal muscle leading to muscle wasting in patients with acute diabetes results from an accelerated Ub conjugation and protein degradation via the N-end rule pathway. The same pathway is activated in cancer cachexia, sepsis and hyperthyroidism (Lecher et al., 1999, J. Clin. Invest., 104: 1411-1420; and Solomon et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 12602-12607).